(1→3)-β-d-glucan as a measure of active mold

ABSTRACT

Electrokinetic devices and methods are described with the purpose of collecting assayable agents from a dielectric fluid medium. Electrokinetic flow may be induced by the use of plasma generation at high voltage electrodes and consequent transport of charged particles in an electric voltage gradient. Actively growing mold releases the carbohydrate cell wall component (1→3)-β-D-Glucan into the air. The invention recognizes that the airborne fraction is that which affects respiratory health and selectively tests for a free form which is soluble in aqueous medium. The sample to be analysed is preferably collected by the electrokinetic propulsion method described, but any air sampling method such as filtration, impactor or impingement may be applicable.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the collection of and sampling ofassayable agents in a dielectric medium. This includes, but is notlimited to, sampling air for agents whose presence or absence isdeterminable by bio-specific assays for mold or mold spore cell wallcomponents. The field includes sampling of air for biological agents,direction to, and deposition on, a collection means for an assay device.The agent-specific assays may include immunoassays or chromogenic assaysbased on proteolytic pathways. Assays may include, but are not limitedto, detection means which are colorometric, fluorescent, turbidimetric,electrochemical or voltammetric.

Description of the Prior Art

(1→3)-β-d-glucan, like allergens (1-3) are present in spores but arereleased into air by germinating and growing fungi in particulate formnot readily recognizable microscopically as spores. Thus, while(1→3)-β-d-glucan itself is not an allergen, the two are likely to beassociated in the air. (1→3)-β-d-glucan is also found in pollen (4).

Structure:

(1→3)-β-D-glucan are non-allergenic water-insoluble structural cell wallcomponents of most fungi, some bacteria, most higher plants and manylower plants. Glucans may account for up to 60% of the dry weight of thecell wall of fungi, of which the major part is (1→3)-β-D-glucan (Klis,1994). They consist of glucose polymers with variable molecular weightand degree of branching i.e. triple helix, single helix or random coilstructures (Williams, 1997). In the fungal cell wall, (1→3)-β-D-glucansare linked to proteins, lipids and carbohydrates such as mannan andchitin, and they contain (1→3)-b-glucan side-branches which may connectwith adjacent (1→3)-β-D-glucan polymers.

Relation to Symptoms:

Levels in air correlate with fungal mass and severity of symptoms. Thisis not a causal relationship because of contribution of allergensreleased from fungi (5). B-glucan correlates with Peak Respiratory FlowVariability (PFV) in children with 50% having pre-existing respiratoryconditions (6).

Douwes reviewed literature pertaining to B-glucan and respiratory health(7). They concluded the studies reviewed suggested some associationbetween (1→3)-β-D-glucan exposure, airway inflammation and symptoms.Potential underlying inflammatory mechanisms associated with exposurecould not be identified. Population studies and animal and human studiesusing experimental exposure are included.

Assays:

Three kinds of assay have been used in the literature: inhibitionimmunoassay immunoassay (8), sandwich monoclonal immunoassay (9) andlimulus amebocyte assay. The latter is commercialized by Associates ofCape Cod. Brooks et al (10) did an inter-laboratory comparison of the 3methods. Results obtained with different methods were oftensignificantly correlated and therefore comparable in relative terms, butdirect comparison of results between laboratories and assays isinappropriate.

Extraction:

Most β-(1,3)-glucans are water insoluble at ambient temperature, and twoextraction methods either alkali extraction (typically vortex followedby 1 hr shaking in 0.3N NaOH) and hot water extraction (typically 0.05%TWEEN® 20 (TWEEN is a registered trademark of CRODA AMERICAS LLC) inPBS, end-over-end rotation, autoclave 60 min vortex). Alkalineextraction has been used in limulus amebocyte-based methods, and heatextraction with EIA-based methods. Extraction methods used are reviewedin (10). Measurements on spores from filters (11) were done includingtreatment with 0.15N NaOH. Followed by neutralization with equal volumetris-CI at twice the normality of the NaOH. Not all papers mention theneutralizations step. No extraction was mentioned in the pollenpublication (4).

Madsen et al described optimization of extraction with NaOH (12) andreviewed some work where extraction is or is not used or not mentioned.No controls were done in this or other work with no extraction. It isfrequently stated that the extraction is required to solubilize the(1→3)-β-D-glucan.

Size Range:

Foto et al did a study with extensive samples from damaged homes (13).They state that (1→3)-β-D-glucan is associated with fragments, hyphae aswell as spores. No actual direct data was given. Lee et al (14). did anindoor to outdoor comparison of (1→3)-β-D-glucan. They also mentionfragments but provide no data. Saleres et al (15). found in airbornefraction a smaller size range than intact spores, which, they say,implies deeper lung penetration. Madsen et al (16) found a largefraction of (1-3)-β-D-glucan in the PM1 fraction which contained nospores. In spite of the small size, they still say material was “madewater soluble” using 0.3M NaOH.

Singh et al (17, 18) did size fractionation with the NIOSH sampler, andfound equal amounts (1→3)-β-D-glucan in 3 size cuts going down to PM1fraction.

Chen et al compared inside v. outside and effect of human activity (19,20). They found the smallest size was least affected by human activitybut more (1-3)-β-D-glucan in larger size particles.

Surrogate Assay:

Vogelmark et al (21) did measurements on Penicillium, Aspergillus,Stachrybotis cultures for spores. They do not mention any extractionmethod. They recommend (1→3)-β-D-glucan measurement as surrogate formold exposure.

Chew et al (22) used vacuumed dust extract (22)(21) and did correlationof (1→3)-β-D-glucan with viable spores. They say dust is a surrogate forairborne exposure.

Speciation:

Iossifova et al compared with mold species in dust using EPA standardmultiplex qPCR for molds (23). There was no correlation between(1→3)-β-D-glucan with Relative Moldiness Index (RMI) derived from thefrom the qPCR. They used complicated multivariate statistical analysisto show correlation with the predominant species. Cladosporium andAspergillus genera, as well as Epicoccum nigrum, Penicilliumbrevicompactum and Wallemia sebi. contributors There was no correlationwith Alternaria. This does not mean Alternaria produces no(1→3)-β-D-glucan.

LIST OF REFERENCES

-   1. Green B J, Schmechel D, Summerbell R C. Aerosolized fungal    fragments. Adan O C G, Samson R A, editors. Wageningen: Wageningen    Acad Publ; 2011.-   2. Green B J, Tovey E R, Sercombe J K, Blachere F M, Beezhold D H,    Schmechel D. Airborne fungal fragments and allergenicity. Medical    Mycology. 2006 September; 44:S245-S55.-   3. Green B J, Mitakakis T Z, Tovey E R. Allergen detection from 11    fungal species before and after germination. J Allergy Clin Immunol.    [Article]. 2003 February; 111(2):285-9.-   4. Rylander R, Fogelmark B, McWilliam A, Currie A.    (1->3)-beta-D-glucan may contribute to pollen sensitivity. Clin Exp    Immunol. 1999 March; 115(3):383-4.-   5. Rylander R. Indoor air-related effects and airborne    (1->3)-beta-D-glucan. Environ Health Perspect. 1999 June; 107:501-3.-   6. Douwes J, Zuidhof A, Doekes G, van der Zee S, Wouters I, Boezen H    M, et al. (1->3)-beta-D-glucan and endotoxin in house dust and peak    flow variability in children. American Journal of Respiratory and    Critical Care Medicine. [Article]. 2000 October; 162(4):1348-54.-   7. Douwes J. (1->3)-beta-D-glucans and respiratory health: a review    of the scientific evidence. Indoor Air. 2005 June; 15(3):160-9.-   8. Douwes J, Doekes G, Montijn R, Heederik D, Brunekreef B.    Measurement of beta (1->3)-glucans in occupational and home    environments with an inhibition enzyme immunoassay. Applied and    Environmental Microbiology. 1996 September; 62(9):3176-82.-   9. Sander I, Fleischer C, Borowitzki G, Bruning T, Raulf-Heimsoth M.    Development of a two-site enzyme immunoassay based on monoclonal    antibodies to measure airborne exposure to (1->3)-beta-D-glucan.    Journal of Immunological Methods. 2008 August; 337(1):55-62.-   10. Brooks C R, Siebers R, Crane J, Noss I, Wouters I M, Sander I,    et al. Measurement of beta-(1,3)-glucan in household dust samples    using Limulus amebocyte assay and enzyme immunoassays: an    inter-laboratory comparison. Environmental Science-Processes &    Impacts. 2013; 15(2):405-11.-   11. Foto M, Plett J, Berghout J, Miller J D. Modification of the    Limulus amebocyte lysate assay for the analysis of glucan in indoor    environments. Anal Bioanal Chem. 2004 May; 379(1):156=62.-   12. Madsen A M, Frederiksen M W, Allermann L, Peitersen J H.    (1->3)-beta-D-glucan in different background environments and    seasons. Aerobiologia. [Article]. 2011 June; 27(2):173-9.-   13. Foto M, Vrijmoed L L P, Miller J D, Ruest K, Lawton M, Dales    R E. A comparison of airborne ergosterol, glucan and Air-O-Cell data    in relation to physical assessments of mold damage and some other    parameters. Indoor Air. 2005 August; 15(4):257-66.-   14. Lee T, Grinshpun S A, Kim K Y, Iossifova Y, Adhikari A,    Reponen T. Relationship between indoor and outdoor airborne fungal    spores, pollen, and (1->3)-beta-D-glucan in homes without visible    mold growth. Aerobiologia. 2006 September; 22(3):227-36.-   15. Salares V R, Hinde C A, Miller J D. Analysis of Settled Dust in    Homes and Fungal Glucan in Air Particulate Collected during HEPA    Vacuuming. Indoor and Built Environment. [Article]. 2009 December;    18(6):485-91.-   16. Madsen A M, Schlunssen V, Olsen T, Sigsgaard T, Avci H. Airborne    Fungal and Bacterial Components in PM1 Dust from Biofuel Plants. Ann    Occup Hyg. 2009 October; 53(7):749-57.-   17. Singh U, Reponen T, Cho K J, Grinshpun S A, Adhikari A, Levin L,    et al. Airborne Endotoxin and beta-D-glucan in PM1 in Agricultural    and Home Environments. Aerosol and Air Quality Research. 2011    August; 11(4):376-86.-   18. Singh U, Levin L, Grinshpun S A, Schaffer C, Adhikari A,    Reponen T. Influence of home characteristics on airborne and    dustborne endotoxin and beta-D-glucan. J Environ Monit. [Article].    2011 November; 13(11):3246-53.-   19. Chen Q, Hildemann L M. The Effects of Human Activities on    Exposure to Particulate Matter and Bioaerosols in Residential Homes.    Environ Sci Technol. 2009 July; 43(13):4641-6.-   20. Chen Q, Hildemann L M. Size-Resolved Concentrations of    Particulate Matter and Bioaerosols Inside versus Outside of Homes.    Aerosol Sci Technol. 2009; 43(7):699-713.-   21. Fogelmark B, Rylander R. (1->3)-beta-D-glucan in some indoor air    fungi. Indoor and Built Environment. 1997 September-October;    6(5):291-4.-   22. Chew G L, Douwes J, Doekes G, Higgins K M, van Strien R,    Spithoven J, et al. Fungal extracellular polysaccharides, beta    (1->3)-glucans and culturable fungi in repeated sampling of house    dust. Indoor Air-International Journal of Indoor Air Quality and    Climate. 2001 September; 11(3):171-8.-   23. Iossifova Y, Reponen T, Sucharew H, Succop P, Vesper S. Use of    (1-3)-beta-D-glucan concentrations in dust as a surrogate method for    estimating specific fungal exposures. Indoor Air. 2008 June;    18(3):225-32.

U.S. Pat. No. 5,266,461, Tanaka, Method for determining(1→3)-β-D-glucan, is the only patent for assay of (1→3)-β-D-glucan thatwe are aware of. They use an antibody to inhibit the pathway forendotoxin sensitive factor.

In the prior art, there exist many examples of collection of agents fromthe air for bioassay. For example, the following publications describevarious methods of allergen, pathogen and toxin collection for assay:

-   1. Yao et at (2009) in Aerosol Science volume 40, pages 492-502.-   2. Noss et al (2008) in Applied and Environmental Microbiology,    volume 74, pages 5621-5627.-   3. King et al (2007) in Journal of Allergy and Clinical Immunology,    volume 120, pages 1126-31.-   4. Earle et al (2007) in Journal of Allergy and Clinical Immunology,    volume 119, pages 428-433.-   5. Peters et al (2007) in Journal of Urban Health: Bulletin of the    New York Academy of Medicine, volume 84, pages 185-197.-   6. Yao and Mainelis (2006) in Journal of Aerosol Science, volume 37,    pages 513-527.-   7. Platts-Mills et al (2005) in Journal of Allergy and Clinical    Immunology, volume 116, pages 384-389.-   8. Sercombe et al (2004) in Allergy, volume 60, pages 515-520.-   9. Custis et al (2003) in Clinical and Experimental Allergy, volume    33, pages 986-991.-   10. Polzius et al (2002) in Allergy, volume 57, pages 143-145.-   11. Tsay et al (2002) in Clinical and Experimental Allergy, volume    32, pages 1596-1601.-   12. Parvaneh et al (2000) in Allergy, volume 55, pages 1148-1154.-   13. McNerney et al (2010) in BMC Infectious Diseases, volume 10,    pages 161-166 and device in U.S. Pat. No. 7,384,793.

Other known methods of sample collection include trapping of volatileorganic compounds (VOC) on activated carbon, de-sorption and analysis bymass spectrometry. See Phillips et al (2010) in Tuberculosis, volume 90,pages 145-151 and references therein. VOC's are not consideredencompassed by the present invention since the assays are strictlychemical in nature, and are not bio-specific as defined here. Bybio-specific is meant assays wherein the result is determined by abiological specificity such as nucleic acid specificity, antibodyspecificity, receptor-ligand specificity and the like. While diagnosticspecificity may be achieved by VOC analysis, this is inferred bypresence and amount of groups of defined organic compounds.

The foregoing prior art publications describe “dry” methods usingpumping and filtration, wiping, passive deposition, electrokinetictransport etc; usually followed by an extraction step and application ofthe extract to an assay.

Methods for collection in a liquid stream have been described in thepatent literature:

-   Yuan and Lin in US Patent Application 2008/0047429A1.-   Saski et al in U.S. Pat. No. 6,484,594 issued in 2002.

While efficiently collecting agents from the air, such liquid streamingsystems inevitably result in high dilution of the sample. There is aconsequent trade-off in sensitivity unless the agents arere-concentrated.

Northrup et al in U.S. Pat. Nos. 7,705,739 and 7,633,606 describe anautonomously running system for air sampling and determination ofairborne substances therein. They do not specify the exact method of airsampling, nor detail how it is transferred to an assay system.

There exist numerous commercially available systems for air purificationbased on filtration or electrostatic precipitation. For a generaldescription see the Environmental Protection Agency article “Guide toAir Cleaners in the Home”, U.S. EPA/OAR/ORIA/indoor EnvironmentsDivision (MC-6609J) EPA 402-F-08-004, May 2008. Numerous commercialexamples of systems exist using either High Efficiency Particulate Air(HEPA) filters or electrostatic precipitation filters. Such systems arewidely used for removal of particulate matter or allergens from air,including as part of domestic heating, ventilation and air conditioning(HVAC) systems. HEPA filters have the advantage of removal of particlesdown to the micron size range, whereas electrostatic precipitationmethods have the advantage of entailing high volume flow with little orno pressure differential. See US patent by Bourgeois, U.S. Pat. No.3,191,362 as a detailed example for the technical specification of anelectrostatic precipitation system. While efficiently removing agentsfrom the air, such air purification systems do not lend themselves tocollection of samples for analysis.

Filtration methods are well-known for collection of air samples fortesting. Such filtration methods may also be used for capturingparticles containing (1→3)-β-D-glucan for assay.

Electrokinetic-based air cleaning systems have been developed andformerly commercialized by the company Sharper Image (but nowdiscontinued) under the trade name Ionic Breeze. The originalelectrokinetic principle was enunciated by Brown in U.S. Pat. No.2,949,550. This was further improved by Lee in U.S. Pat. No. 4,789,801for improving airflow and minimizing ozone generation. Furtherimprovements for the commercially available system are described in USpatents by Taylor and Lee, U.S. Pat. No. 6,958,134; Reeves et al, U.S.Pat. No. 7,056,370; Botvinnik, U.S. Pat. No. 7,077,890; Lau et al, U.S.Pat. No. 7,097,695; Taylor et al, 7,311,762. In the foregoingdescriptions of devices using electrokinetic propulsion, a commonelement is a high voltage electrode consisting of a wire. A very steepvoltage gradient is generated orthogonally to the wire because of thevery small cross-sectional area of the wire. The high voltage gradientcauses the creation of a plasma consisting of charged particles, andkinetic energy is imparted to the charged particles by the high voltagegradient. The resulting net air flow is created by exchange of kineticenergy between charged and uncharged particles, and the net air flow isdirected by the juxtaposition of planar electrodes which are at zero oropposite sign voltage to that of the wire electrode. Charged particlesare electrostatically precipitated on to the planar electrodes, whichmay periodically be removed for cleaning. This body of work is directedtoward air purification, not sample collection. However, as firstdescribed by Custis et al (2003), the Ionic Breeze device has beenadapted for sample collection for allergen analysis by wiping down theelectrodes with a paper tissue. The allergens were extracted from thetissue and subject to an immuno-assay. The Ionic Breeze was also used inthe works of Peters et al (2007) and Platts-Mills et al (2005) forallergen collection for immunoassay analysis. Earlier, Parvaneh et al(2000) described an ionizer device with a “metal cup having a conductivesurface as a collector plate”, from which allergens are extracted forassay. It is not evident how the sample is collected on the inside of ametal cup and does not adhere to the entire surface. The device was madeby Airpoint AB, Stockholm, Sweden. However, there is no publicinformation concerning the manufacture or sale of such a product byAirpoint AB, there is insufficient information for one skilled in theart to be able to understand the details of the device, and no similardevice was used by the same authors in subsequent publications onenvironmental allergen detection. There is no mention of focusing of thesample into a potential well created by a voltage gradient.

Yao et al (2009) and Yao and Mainelis (2006) have described methods forcollection of bio-assayable agents on to an assay means or device. Yaoand Manielis (2006) describe blocks of agar gel in electrical contactwith planar electrodes, and Yao et al (2009) describe a microtiter plateinterposed between planar electrodes. Both of these works describe aflow of air driven by a pump, and electrostatically precipitating theagents to be analyzed on to the assay means. The electrodes and the agarblocks have substantially the same area in these works.

McNerney et al (2010) describe a breathalyzer device, where theindividual breathes or coughs into a breathing tube, the sample collectson the internal surface of a tube, is scraped with a plunger on to anoptical biosensor, an immunological binding reaction is performed andthe biosensor utilizes an evanescent wave illumination system todetermine the presence or absence of M. tuberculosis by scattered light.

Inspirotec Inc, the Applicant herein, has commercialized an ion capturedevice for assay of biological materials. This and related devices aredescribed in Inspirotec's U.S. Pat. No. 9,618,431, Electrokinetic devicefor capturing assayable agents in a dielectric fluid; U.S. Pat. No.9,481,904 Electrokinetic method for capturing and bio assaying airborneassayable pathogenic agents; U.S. Pat. No. 8,038,944, Electrokineticdevice for capturing assayable agents in a dielectric fluid; U.S. Pat.No. 9,360,402, Electrokinetic device for capturing assayable agents in adielectric fluid utilizing removable electrodes; and U.S. Pat. No.9,216,421, Integrated system for sampling and analysis). Ion capturetechnology may be used for capturing airborne particles containing(1→3)-β-D-glucan.

None of the prior art suggests use of the soluble fraction of(1→3)-β-D-glucan as a measure of mold exposure.

SUMMARY OF THE INVENTION

(1→3)-β-d-glucan is a structural component of cell walls of molds, butcan also be found in yeast, mold, pollen, bacteria. The largest fractionin the air is attributable to molds.

Assays that have been used are competitive immunoassays, sandwichimmunoassays and limulus amebocyte lysate (LAL) assays. Competitiveimmunoassays are less sensitive, sandwich immunoassays had equivalent toLAL, and were claimed to be less sensitive and more specific. The LALassay depends on the activation of a specific protease from horseshoecrab which creates a fluorescent signal from a synthetic substrate. Theresult is an extraordinarily sensitive assay. The specificity does notappear to have been an issue, and the majority of continuing work hasused the commercially available LAL assay.

There have been many proposals that measurement of (1→3)-β-d-glucan is agood surrogate for total mold exposure.

A correlation was shown between levels of (1→3)-β-d-glucan in house dustand respiratory symptoms by peak expiratory flow (PEF).

In homes with active mold growth, (1→3)-β-d-glucan was found in sizesranging from 18 to 0.18 micron. This means that a large fraction was inparticles and fragments smaller than spores. The smaller range ofparticles are more likely to remain airborne longer and penetrate thelungs deeper, just like dust mite allergens.

As a structural cell wall component, the (1→3)-β-d-glucan is usually inan insoluble form. Therefore, all testing that has been done to date hasused either an extreme heating step or an alkaline treatment to renderit soluble for testing. The fact that a large fraction of the airborneform is in a range where the particles are soluble has escaped notice.We routinely remove all insoluble and particulate material from samplesfrom our device prior to immunoassay. That fraction will contain thelower range of particulate size that will penetrate deeply into thelungs.

Alkaline extraction of this fraction has little effect on results (seeexamples).

Measurement of airborne soluble fraction of (1→3)-β-d-glucan istherefore both a direct measurement of a respiratory irritant whoserelease also parallels the release into the air of fungal allergens.None of the prior art suggests direct measurement of a free form of(1→3)-β-d-glucan in samples collected for airborne material. Measurementof the free form as a soluble fraction is the subject of this invention.This free form will represent smaller size particles which are wellknown to penetrate deeply into the alveoli of the lungs and thus triggerrespiratory problems such as allergic reactions deep in the lungs whichresults in asthma. This measurement is then a parameter that parallelsthe behavior of allergens in general and is therefore a useful surrogatefor assessing exposure to mold allergens in general.

In the preferred embodiment of the invention, the soluble fractioncontaining free (1→3)-β-D-glucan is collected by an electrokineticpropulsion device, for example, as commercialized by the inventors underthe names Inspirotec or Exhale. However, depending on the needs, otherdevices well-known to those skilled in the art, such as filtration ofair through filters with defined pore sizes, such as microporousmembrane filters or fibrous filters, or HEPA filters or Electret filtersas commercialized by the company 3M, or filters depending capture ofdefined size classes of particles by impaction, may be used. Examplesare Anderson Impactors, and the NIOSH cyclone impactor (Lindsley et al,Journal of Environmental Monitoring, 2006, 8, 1136-42) or Spin Con wetcyclonic method or Bobcat electret-based filtration as commercialized byAlburty Labs, of Drexel, Mo.

Other objects, features, and advantages of the invention will becomeapparent from a review of the entire specification, including theappended claims and drawings.

BRIEF DESCRIPTION OF THE INVENTION

FIG. 1 represents a generic electrokinetic flow device.

FIGS. 2A-2D represent an assembled carrier with removable electrodes.

FIG. 3 is a graphical representation of the statistical analysis of acomparison of (1→3)-β-D-glucan compared with presence or absence of moldallergen in those same airborne samples.

FIG. 4 is a graphical representation of the statistical analysis of thecomparison of (1→3)-β-D-glucan determination on a set of airbornefractions treated or not treated with NaOH.

FIG. 5 is a graphical representation of the distribution of(1->3)-β-D-glucan in 76 homes throughout the United States.

DETAILED DESCRIPTION OF THE INVENTION

Air is sampled by a sampling device which maybe as described in detailin prior patents issued to the Applicant, namely U.S. Pat. Nos.8,038,944, 9,216,421, 9,360,402, 9,481,904 and 9,618,431. Thespecification of each of these patents is incorporated by referenceherein. Particular attention is drawn to U.S. Pat. No. 9,360,402 for theanalysis of bio-specific material using a cartridge with removeableelectrodes. This device provides the convenience of extraction of thebio-specific agents by immersing the detached electrodes directly inbuffer extraction medium and shaking in a centrifuge tube with a vortexmixer, as is described in detail below.

Example 1

FIG. 1 represents a basic ion propulsion device with a housing, 1, withhigh voltage wire seen in cross-section, 2, and symbolic representationof voltage contours, grounded plate electrodes 3. Resulting ion flow isrepresented by arrow 4. Resulting net air in-flow is represented byarrow 5 and outflow by arrows 6. Advantageously, the electrodes 3 areremovable and may be mounted to a carrier, which is removable from thehousing 1, as shown in U.S. Pat. No. 9,360,402.

Example 2

FIGS. 2A and 2B illustrates a removable carrier assembly 21 that couldbe used with the housing of FIG. 1 as an alternative to fixedelectrodes. The carrier assembly 21 is described in detail in U.S. Pat.No. 9,360,402. The carrier assembly 21 includes a one-piece plasticcarrier 23, a latch 25 and capture electrodes 27. The carrier 23 isremoveable from the housing 1. Moreover, the capture electrodes 27 areremovably secured to the carrier 23. The latch 25 supports theelectrodes 27, see FIGS. 2C and 2D. The latch 25 is adapted to securethe capture electrodes 27 to the carrier 23. For testing, the carrier 23can be removed from the housing 1. The latch 25 and electrodes 27 canthen be removed from the carrier 23 to facilitate testing.

Example 3

Samples were run in a variety of mold positive and mold negative homeenvironments. The presence of airborne mold allergens were determined bymultiplex immunoassays using MARIA kits from Indoor Biotechnologies andthe MagPix instrument supplied by BioRad Inc. Samplers were routinelyrun for 5 days and the allergens and (1→3)-β-D-glucan containingmaterial extracted from the detached electrodes as described in detailin Example 4, and the supernatants tested by MARIA and by Glucatellassay kit (Associates of Cape Cod Inc., East Falmouth, Mass.) followingmanufacturers protocol for kinetic rate assay.

FIG. 3 shows a statistical analysis of comparison of presence of moldallergens determined by the MARIA™ multiplex immunoassay method for themold allergens Alt a 1 and Asp f 1) and (1→3)-β-D-glucan. The box plotsshow the 90 percentile ranges and the full range of values. The resultssummarized in FIG. 3 show a significant relationship between the absenceof mold allergen and the absence of (1→3)-β-D-glucan in the airbornesamples. A higher mean is also observed in the mold allergen positivegroup (7.19) compared to the negative group (2.42). Data from FIG. 3shows a possible significant relationship between airborne mold allergenand the presence of (1→3)-β-D-glucan.

Example 4

Effect of NaOH extraction on measured (1→3)-β-D-glucan.

TABLE 1 Sample NaOH Treated (fg/L) NaOH Untreated (fg/L) 1 9.49 5.86 221.60 15.28 3 4.75 7.02 4 3.36 3.96 5 4.27 2.05 6 3.83 4.10

The Inspirotec air sampling device, described above, was run for 5consecutive days in each test environment. Following testing, stainlesssteel electrode strips were removed from cartridges, located in thedevice, and transferred to 15 ml centrifuge tubes. One ml of PBS with0.02% TWEEN® 20 was added to the tubes and vortexed intermittently over10 minutes. Samples were removed from the centrifuge tubes andtransferred to 2 mL screw-cap tubes, then centrifuged at 15,000 g for 30minutes. The supernatants were removed and placed in new 2 ml screw-captubes. In another 2 mL screw-cap tube, 80 μl of these samples werebrought to 0.05N NaOH by addition of 20 uL of 2.5N NaOH. Samples wereshaken for 2.5 hrs at room temperature o an orbital shaker, neutralizedby addition of 100 μL of 2M Tris-HCL (1M Tris-HCL final).(1→3)-β-D-glucan levels were determined using the Glucatell assay kit(Associates of Cape Cod Inc., East Falmouth, Mass.) followingmanufacturer's standard protocol for kinetic rate assay.

The results are also shown graphically in FIG. 3 as analyzedstatistically with the JMP package, JMP® Pro 13.0.0 (SAS Institute Inc.Cary, N.C.).

The slope of the best fit straight line is 1.41. This shows that whensamples are collected and analyzed in this manner, 41% of the(1→3)-β-D-glucan is in an insoluble fraction. The current inventionfocuses attention on the soluble fraction, as determined by thesupernatant from centrifugation and no extraction.

Example 5

Distribution of (1→3)-β-D-glucan levels in homes throughout the U.S.

Air samples were collected from 76 homes across the United States, usingthe Inspirotec device. In each home, the device was run for a period of1 to 5 days. After completion of running period, stainless steelelectrode strips were removed from cartridges, located in the device,and transferred to 15 mL centrifuge tubes. One mL of 1×PBS with 0.02%Tween® 20 was added to the tubes and vortexed intermittently over aperiod of 10 minutes. Samples were removed from 15 mL centrifuge tubesand transferred to 2 mL screw-cap tubes. They were then centrifuged at15,000 g for 30 minutes. The supernatants were removed and placed in new2 mL screw-cap tubes. (1→3)-β-D-Glucan concentration was measured usingthe Glucatell assay kit (Associates of Cape Cod Inc., East Falmouth,Mass.) following manufacturers protocol for kinetic rate assay.

The Results are shown graphically in FIG. 4 as analyzed statisticallywith the JMP package, JMP® Pro 13.0.0 (SAS Institute Inc. Cary, N.C.).Values below the mean values of zero time field controls for the assaywere assigned a value of field control/2 and number of occurrences oflog₁₀ of the values were plotted in bins as shown on the x-axis.

(1→3)-β-D-Glucan was detected in 62% of homes.

It is apparent from the foregoing that measurement of (1→3)-β-D-Glucanin the soluble fraction of samples collected from airborne material is arepresentation of the fraction of free (1→3)-β-D-Glucan in the air thatis material released from actively growing molds and will penetrate mostdeeply into the respiratory system with impact on respiratory health.This fraction may also parallel the release of allergens from molds sothat it is also be a surrogate assay for airborne allergen exposure.Previous art ignored the soluble fraction and focused on materialextractable from larger complexes. It will be obvious to those skilledin the art that the soluble fraction may be prepared by the preferredmethod of centrifugation as described here, or by other well knownmethods such as filtration or settling.

Thus, there is described herein a method for analyzing aerosolparticles, wherein said aerosol particles are captured by an airsampling device, extracted from the sampling medium and soluble fractionanalyzed for (1→3)-β-D-glucan. The air sampling device may be based onelectrokinetic propulsion or on electrostatic precipitation.(1→3)-β-D-glucan may be determined by a limulus-amebocyte based assay orby an immunoassay. The sampling device may be based on filtration, onimpingement, or on an impactor.

There is also described a method for analyzing aerosol particles,wherein said aerosol particles are deposited on an electrode of anelectrokinetic propulsion device from a volume of air propelledelectrokinetically through said device, said electrode being removablyattached to a carrier mounting; said electrode is removed from saidcarrier mounting and placed in an extraction vessel; a predeterminedvolume of extraction fluid is added to said extraction vessel; saidelectrode is agitated in said extraction fluid for a predetermined time;and all or part of said extraction fluid is added to a reaction mixturefor analysis of said aerosol particles for (1→3)-β-D-glucan.

What we claim is:
 1. A method for analyzing aerosol particles,comprising capturing aerosol particles using an air sampling device,suspending said captured aerosol particles from the air sampling devicein a extraction fluid, performing centrifugation of said suspension,directly adding with no other processing all or part of supernatant fromsaid centrifugation to a reaction mixture for analysis of saidsupernatant for (1→3)-β-D-glucan.
 2. A method according to claim 1wherein the air sampling device is based on electrokinetic propulsion.3. A method according to claim 1 where the sampling device is based onelectrostatic precipitation.
 4. A method according to claim 1 comprisingdetermining (1→3)-β-D-glucan level by a limulus-amebocyte based assay.5. A method according to claim 1 comprising determining (1→3)-β-D-glucanlevel by an immunoassay.
 6. A method according claim 1 wherein thesampling device is based on filtration.
 7. A method according claim 1wherein the sampling device is based on impingement.
 8. A methodaccording claim 1 wherein the sampling device is based on an impactor.9. A method for analyzing aerosol particles, comprising: capturingaerosol particles on an electrode of an electrokinetic propulsion devicefrom a volume of air propelled electrokinetically through said device,said electrode being removably attached to a carrier mounting; removingsaid electrode from said carrier mounting and placing said electrode inan extraction vessel; adding a predetermined volume of extraction fluidto said extraction vessel; agitating said electrode in said neutralextraction fluid for a predetermined time; and directly adding with noother processing all or part of said neutral extraction fluid to areaction mixture for analysis of said aerosol particles for(1→3)-β-D-glucan.
 10. A method according to claim 9 comprisingdetermining (1→3)-β-D-glucan level by a limulus-amebocyte based assay.11. A method according to claim 9 comprising determining(1→3)-β-D-glucan level by an immunoassay.